The chromatin structure of the beta-globin gene locus is dynamic. In erythroid cells, the chromatin structure of the locus undergoes a dramatic reorganization over approximately 2OOkb of DNA producing at least three erythroid-specific chromatin structures. This reorganization is thought to involve the """"""""opening-up"""""""" of chromatin to allow trans-acting factors to interact with regulatory elements. Understanding how these domains of altered chromatin structure are created is central to understanding the complex mechanisms by which globin gene expression is regulated. We hypothesize that there are specific functional elements within the beta- globin locus control region (LCR) which direct the formation of the erythroid-specific chromatin structures of the human beta-globin gene locus. Our long-term goals involve the identification and characterization of these functional elements. Just as assays of gene expression have been used to identify and characterize the active elements of globin promoters, enhancers, and LCR domains, we propose to use assays of chromatin structure to locate, and then characterize, the specific functional elements responsible for the formation of the chromatin structures of the globin locus. Specifically, experiments are planned to continue our characterization of an element which is necessary for the formation of the unique erythroid-specific structure of a DNase 1 HS of the LCR. Other experiments are designed to assess the role of the erythroid-specific chromatin structures in the regulation of globin gene expression. We hypothesize that the transcription factors GATA-1 and NF-E2 are the key factors in forming the erythroid specific chromatin structures of the globin locus. We will test this hypothesis. Experiments are also proposed to locate the boundaries of the globally-altered chromatin structure which characterizes the beta-globin locus. The original impetus for our studies of the chromatin structure of the beta-globin gene locus was to better understand how an """"""""active"""""""" chromatin structure is established surrounding the genes of the locus in erythroid cells. These studies were initiated as part of a larger effort to develop more efficient strategies for gene therapy of severe beta-thalassemia and sickle cell disease. One problem which has hampered the development of gene therapy for these conditions has been an inability to achieve consistent high-level, tissue-specific, expression of transferred gene constructs. We hope that by identifying of the functional elements which form the domains of active chromatin structure around the native beta- globin genes we will be able to incorporate these elements into future therapeutic constructs leading to clinically applicable globin expression vectors.
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